Beginning with the discovery of BCL-2 at the t14;18 chromosomal breakpoint of follicular lymphoma1-3, the anti-apoptotic members of the BCL-2 family have emerged as key pathogenic proteins in human diseases characterized by unchecked cellular survival, such as cancer and autoimmunity. A series of anti-apoptotic proteins including BCL-2, BCL-XL, BCL-w, MCL-1, BFL1/A1, and BCL-B promote cellular survival by trapping the critical apoptosis-inducing BCL-2 homology domain 3 (BH3) α-helix of pro-apoptotic BCL-2 family members4. Cancer cells exploit this physiologic survival mechanism through anti-apoptotic protein overexpression, establishing an apoptotic blockade that secures their immortality. To overcome this potentially fatal resistance mechanism, a pharmacologic quest is underway to develop targeted therapies that bind and block BCL-2 family survival proteins.
Anti-apoptotic proteins contain a hydrophobic binding pocket on their surface that engages BH3 α-helices4,5. Because Nature's solution to anti-apoptotic targeting involves selective interactions between BH3 death domains and anti-apoptotic pockets6,7, molecular mimicry of the BH3 α-helix has formed the basis for developing small molecule modulators of anti-apoptotic proteins8-10. Promising compounds undergoing clinical evaluation, such as ABT-26311, obatoclax9, and AT-10112, each target three or more anti-apoptotic proteins. The development of more precise inhibitors that target individual anti-apoptotic proteins remains a significant challenge due to the often subtle differences among BH3-binding pockets. Reminiscent of the long-term goals in kinase therapeutics, anti-apoptotic inhibitors with greater specificity would provide finely-tuned therapies to treat distinct diseases while potentially avoiding unwanted side-effects, such as those observed for ABT-26311 and AT-10113. In addition, such compounds would serve as invaluable research tools to dissect the differential biological functions of anti-apoptotic proteins.
The specificity of anti-apoptotic proteins for BH3 domains is conferred by the topography of the canonical binding groove and the distinctive amino acid composition of the interacting BH3 helix. Whereas some BH3 domains, such as that of pro-apoptotic BIM, can tightly engage all anti-apoptotic pockets, others are more selective like the BAD BH3 that binds BCL-2, BCL-XL, and BCL-w and the NOXA BH3 that targets MCL-1 and BFL1/A16. The differential binding capacity of BH3 domains and their mimetics is clinically relevant, as exemplified by the close relationship between inhibitor binding spectrum and biological activity. For example, ABT-737, the prototype small molecule BH3 mimetic modeled after the BH3 domain of BAD, was designed to specifically target BCL-2 and BCL-XL10, and induces apoptosis in select cancers that are driven by these proteins14-16. However, ABT-737 fails to show efficacy against cancer cells that overexpress MCL-1, as this anti-apoptotic lies outside the molecule's binding spectrum15-17. In an effort to overcome the challenge of designing precision small molecules to selectively target interaction surfaces that are comparatively large and more complex, we investigated whether Nature's selective BH3 domains could be used to rapidly identify precise small molecule modulators.
The development of precise inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathologic cell survival remains a formidable but pressing challenge. Such compounds would provide finely-tuned molecular probes to study and treat human diseases driven by specific anti-apoptotic blockades. For example, anti-apoptotic MCL-1 has emerged as a major resistance factor in cancer.
MCL-1 overexpression has been linked to the pathogenesis of a variety of refractory cancers, including multiple myeloma18,19, acute myeloid leukemia16, melanoma20, and poor prognosis breast cancer21. MCL-1 exerts its pro-survival activity at the mitochondrial outermembrane where it neutralizes pro-apoptotic proteins such as NOXA, PUMA, BIM, and BAK. The critical role of MCL-1 in selective apoptotic resistance has been highlighted by the sensitizing effects of small interfering RNAs that downregulate MCL-1 protein levels22,23.
Despite the formidable challenges associated with developing precise small molecule modulators of biomedically relevant protein targets, the identification of novel and selective small molecules modulators of MCL-1 is described herein.